Abstract Klebsiella pneumoniae is a persistent human pathogen that is responsible for severe healthcare-associated infections. The bacterium is commonly found associated with indwelling medical devices and hospital surfaces due to its ability to form a resilient biofilm. In susceptible patients, chronic carriage profoundly complicates hospitalization procedures, necessitating enhanced isolation measures to prevent the dissemination of the pathogen in healthcare environments. All of this is ongoing during the emergence of Carbapenem-resistant Enterobacteriaceae (CRE) that are unaffected by most available antibiotics. Both the Centers for Disease Control and the NIH have concluded that this situation poses an urgent threat to public health, and must be immediately addressed via development of new therapies. This proposal focuses on the main virulence factor that K. pneumoniae possesses: robust production of a polysaccharide capsule that allows it to efficiently colonize patients. The synthesis of this capsule is regulated by the Rcs phosphorelay, a multicomponent signaling pathway conserved in all Enterobacteriaceae that involves at least one essential gene. Previous knowledge of this pathway gained in E. coli will inform the analysis of Rcs in K. pneumoniae. E. coli and K. pneumoniae Rcs component structures will be investigated with the help of collaborating crystallographers, and downstream gene regulation will be assessed in both organisms. In addition, an in- vivo fluorescent reporter assay has been developed to examine the ability of small molecules to interfere with the Rcs phosphorelay. This will facilitate novel antibiotic design against Rcs in a high-throughput format. Finally, genetic investigation in K. pneumoniae is relatively primitive. Development of efficient genetic tools in this bacterium is necessary to make chromosomal mutations and to create appropriate reporter constructs to assess the biology of the Rcs pathway as drug target. Any progress made in Klebsiella genome editing using CRISPR or recombineering will be a benefit to the scientific community. These studies will inform the design of novel antibiotics against virulent, drug resistant Enterobacteriaceae. This proposal addresses a central part of the mission of NIH: to both evaluate bacteria for new drug targets and to create the next generation of antimicrobials.